CN101588821A - Oligonucleotide non-viral delivery systems - Google Patents

Oligonucleotide non-viral delivery systems Download PDF

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CN101588821A
CN101588821A CNA2007800343442A CN200780034344A CN101588821A CN 101588821 A CN101588821 A CN 101588821A CN A2007800343442 A CNA2007800343442 A CN A2007800343442A CN 200780034344 A CN200780034344 A CN 200780034344A CN 101588821 A CN101588821 A CN 101588821A
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sirna
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P·A·S·阿尔图松
M·M·伊萨
S·P·斯特兰德
K·M·瓦姆
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FMC Biopolymer AS
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Abstract

Low molecular weight low molecular weight chitosan oligomers were able to self- assemble siRNA into nanosized particles, provide protection against enzymatic degradation, and mediate gene silencing that is stable over a long period of time in vitro. The control of structural variables in formulating complexes of siRNA with low molecular weight chitosans provides an efficient alternative delivery system for siRNA in vitro and in vivo.

Description

Oligonucleotide non-viral delivery systems
Invention field
The present invention generally relates to delivery of nucleic acids and field of gene expression.Specifically, the present invention relates to be used for oligonucleotide, especially the new non-viral delivery systems of siRNA (siRNA).
Background of invention
It is a kind of natural mechanism [1] that relates to by double-stranded short interfering rna (siRNA) specificity downward modulation expression of target gene that RNA disturbs (RNAi).RNAi becomes the common tool [2,3] in functional genomics and target sieving and the external checking more and more.More importantly, be hopeful to find the therapy [4-7] of complex disease such as diabetes, cancer and viral infection based on the exploitation of the medicine of siRNA.For other forms of nucleic acid such as plasmid DNA (pDNA), serum stability is poor, and pharmacokinetics character and invalid cellular uptake remain the main challenge [8] of the gene silencing application of success in the disadvantageous body.A kind of scheme has partly overcome these problems, and this scheme has been utilized the siRNA[9-11 of the chemical modification of tolerance nuclease degradation and cellular uptake improvement].Another kind of scheme relates to the siRNA preparation of utilization based on polycation.For example, based on the preparation of cation lipid effectively [12-15] in the sending of siRNA in vitro and in vivo.On the contrary, think that at first cationic polymer is not suitable for use in oligonucleotide delivery [16].Yet, discover that recently cationic polymer such as polymine (PEI), the tree-shaped polymer of polyamidoamine (PAMAM) and poly-L-Lysine (PLL) can be used for siRNA and send [17-19].The cationic polymer of preparing and sending under the pDNA optimal conditions has opposite report as the efficient of oligonucleotide delivery system.And the toxicity in vivo that some reports are paid close attention to above-mentioned polycation may hinder its following clinical practice [20-22].Therefore, excited the research that is used for the nontoxic effective carrier that siRNA sends.
Summary of the invention
The present invention relates to a kind of compositions, it comprises the complex of following composition: (a) number-average degree of polymerization (DP n) be the low-molecular weight chitoglycan of 30-300, wherein the degree of deacetylation of low-molecular weight chitoglycan is greater than 90%; (b) oligonucleotide.Compositions as claimed in claim 1, said composition comprise the low-molecular weight chitoglycan that utilizes chemistry or enzyme method to be obtained by high molecular weight chitosan.The degree of deacetylation of low-molecular weight chitoglycan is greater than 95%, most preferably greater than 99%.In addition, the net charge of compositions ratio just is essentially.Described low-molecular weight chitoglycan carries out derivatization with targeting aglucon and stabilizing agent.Oligonucleotide comprises the reticent sequence of expressing its function when introducing host cell.Oligonucleotide is selected from: RNA molecule, antisense molecule, ribozyme and Microrna.The pH of the present composition is 3.5-8.0, more preferably 7.1-7.6.The invention still further relates to the method for preparing the present composition, this method may further comprise the steps: low-molecular weight chitoglycan is contacted with aqueous solvent; (b) aqueous solution with step (a) mixes in aqueous solvent with oligonucleotide; (c) keeping the pH of compositions is 3.5-8.0, more preferably 7.1-7.6.The invention still further relates to the preparation method for compositions, this method is included in step (b) afterwards, and the volume that reduces step (b) products therefrom solution is to realize required composition concentration.The invention still further relates to and give mammiferous method with nucleic acid, this method comprises to be utilized described compositions and said composition is introduced in the mammal.Compositions is introduced the intravital method of mammal can be given mucosal tissue by pulmonary, nasal cavity, oral, eyes, buccal, Sublingual, part, rectum or vaginal approach and realize.Perhaps, compositions can by in intravenous, intramuscular, intradermal, intracranial, the canalis spinalis, subcutaneous or intracardiac parenteral approach gives submucous tissue, or give other body surfaces of exposing during internal organs, blood vessel or the surgical operation or body cavity and introduce mammal.The inventive method comprises and gives mammal with described compositions that wherein said oligonucleotide can be at least a its function of mammiferous cell inner expression.The invention still further relates to the using method of the described preparation of compositions that is prepared into preventative or the mammiferous medicine of therapeutic treatment.These application include but not limited to: be used for gene therapy, antisense therapy or the gene vaccine inoculation of preventative or therapeutic treatment malignant tumor, autoimmune disease, hereditary disease, pathogen infection and other pathology diseases.And, the invention still further relates to and utilize described compositions as the diagnostic reagent that uses in the body and in the in-vitro diagnosis method.
Brief Description Of Drawings and sequence table
Fig. 1 has shown the physical stability (A) of siRNA preparation and the protective effect (B) of RNA enzyme A.Use line style DP nThe siRNA complex of 85 chitosans preparation shows the highest physical stability under the condition of the charge ratio of two kinds of pH value and all tests.The polycation of all selections can protect siRNA to avoid the degraded of RNA enzyme A.For agarose gel electrophoresis, 100ng siRNA is loaded in each hole.For chitosan DP n18 preparations, complex is prepared with the charge ratio of 30: 1 (+/-) and 60: 1 (+/-).Then adopted charge ratio 15: 1 (+/-) for the PEI preparation, the complex of siRNA and lipofection amine reagent 2000 is then with weight ratio 2: 1 (+/-) preparation.The representative gel that has shown three independent experiments.
Fig. 2 has shown that (B, C) middle the external of siRNA sent at the HEK293 cell (293-Luc) of normal HEK 293 cells (A) and stably express luciferase.Compare with the contrast untreated cell, realized significant luciferase silence during with specific siRNA-Luc and pLuc (A) or pGFP (C) cotransfection.Luciferase suppresses efficient reduction (B) when only siRNA-Luc being delivered to the 293-Luc cell.48 hours analysis luciferase genes are expressed after the transfection.
Utilize and made chitosan (the branching DP that optimizes for pDNA sends n34) [27] form complex with siRNA with charge ratio preparation in 10: 1.Adopted charge ratio 5: 1 (+/-) for the PEI complex, the complex of siRNA and LF2000 is then prepared with weight ratio 2: 1 (+/-).Gene expression results is expressed as meansigma methods ± S.D.; N=4.
Fig. 3 has shown in the 293-Luc cell with oligonucleotide and has formed the structural change of the line style (A) of complex and branching (B) chitosan to the active influence of luciferase silence.Though as if for chain length chain length and charge ratio greater than the complex that the line style chitosan of 34 monomeric units (number-average degree of polymerization is higher than 34 monomeric units) forms is not crucial, the branching chitosan complexes mediates reticent requirement of effective luciferase has higher charge density and higher charge ratio for the chitosan chain length of length.Adopt siRNA concentration 150 nanomoles (100 nanograms/hole).48 hours analysis luciferase genes are expressed after the transfection.With charge ratio 30: 1 (+/-) preparation chitosan complexes.Gene expression results is expressed as meansigma methods ± S.D.; N=4.
Fig. 4 has shown the siRNA concentration dependent and the relative efficiency of line style low-molecular weight chitoglycan.For lower siRNA concentration (15-30 nanogram/hole is equivalent to 22-44 nanomole/hole siRNA concentration), line style DP nIt is the most high-effect that 85 low-molecular weight chitoglycans show, is about to luciferase expression and strikes and subtract 72-95% with respect to the contrast 293-Luc cell that is untreated.48 hours analysis luciferase genes are expressed after the transfection.With charge ratio 30: 1 (+/-) preparation chitosan complexes.Gene expression results is expressed as meansigma methods ± S.D.; N=4.
Fig. 5 has shown that serum is to the reticent active influence of luciferase.In the various polycation preparations of test, when in transfection medium, having 10% serum, in the 293-Luc cell, all line style chitosans and branching DP n85 keep the reticent activity of its luciferase.Adopt siRNA concentration 150 nanomoles.48 hours analysis luciferase genes are expressed after the transfection.For low-molecular weight chitoglycan and PEI, respectively with charge ratio 30: 1 and 15: 1 (+/-) preparation siRNA complex.The complex of siRNA and LF2000 is with weight ratio 2: 1 (+/-) preparation.Gene expression results is expressed as meansigma methods ± S.D.; N=4.
Fig. 6 has shown the cytotoxicity of siRNA preparation.With measuring dehydrogenase activity (Cytotoxic measurement) in the born of the same parents by the MTT method immediately or after 24 hours behind the various siRNA preparation transfection 293-Luc cells.Opposite with PEI and LF2000, use line style DP nThe interior dehydrogenase activity of cellular morphology and born of the same parents all keeps after the siRNA complex transfection of 85 preparations.Though the LF2000 complex shows significant toxicity immediately after transfection, transfection is the cell viability recovery after 24 hours.Adopt siRNA concentration 150 nanomoles.For low-molecular weight chitoglycan and PEI, respectively with charge ratio 30: 1 and 15: 1 (+/-) preparation siRNA complex.The complex of siRNA and LF2000 is with weight ratio 2: 1 (+/-) preparation.Gene expression results is expressed as meansigma methods ± S.D.; N=4-5.
Fig. 7 shown in external 293-Luc (A, B, C) and the SKOV-3 cell (D) of stably express luciferase in the time-histories of luciferase silence.In 293-Luc and SKOV-3-Luc cell, DP n85 line style chitosan in onset early and show the reticent kinetics of best luciferase aspect the luciferase silence that continues (continuing 5 days) is pointed out stable release in the complete siRNA born of the same parents.Adopt siRNA concentration 44 nanomoles (30 nanograms/hole).For low-molecular weight chitoglycan and PEI, respectively with charge ratio 30: 1 and 15: 1 (+/-) preparation siRNA complex.The complex of siRNA and LF2000 is with weight ratio 2: 1 (+/-) preparation.Gene expression results is expressed as meansigma methods ± S.D.; N=4.
The sequence table summary
SEQ ID NO.1:siGL3 (justice, 5 '-CUUACGCUGAGUACUUCGAdTdT-3 ';
SEQ ID NO.2: antisense, 5 '-UCGAAGUACUCAGCGUAAGdTdT-S1) be the siRNA duplex of the unmodified of targeting luciferase genes (siRNA-Luc), (MedProbe, Lund Sweden) order [28] from the medical probe company of the blue moral of Sweden.
SEQ ID NO.3: mispairing siRNA; The non-targeting siRNA#1 of siCONTROL (siCON1; Justice, 5 '-UAGCGACUAAACACAUCAAUU-3 ';
SEQ ID NO.4: antisense, 5 '-UUGAUGUGUUUAGUCGCUAUU-3 ') (Lafayette CO) orders for Dharmacon Research, Inc. from the DR company limited of state of Colorado Lafayette.
Detailed Description Of The Invention
The line style binary polysaccharide of the shitosan 2-amino that to be a class connected by (1-4)-2-deoxidation-β-D-Glucose (GIcN) and N-acetylation analog 2-acetylaminohydroxyphenylarsonic acid 2-deoxidation-β-D-Glucose (GIcNAc) formation; be biocompatible cationic polymer, it has shown and has been applicable to plasmid pDNA gene delivery [23-27]. Gene delivery can utilize molecular weight distribution fully definite line style and branching shitosan oligomer to realize [25,27] fast and effectively in vitro and in vivo. Have the physical property of improvement based on the compound of shitosan oligomer, comprise that reduced viscosity and polymerization tendency is lower. These compounds also have the efficient of improvement, comprise cellular uptake, onset morning and the high-level gene expression in vivo of improvement.
In the present invention, realized the chitosan of potential low-molecular-weight (7-17kDa) deacetylated substantially fully (deacetylated) in the novel siRNA delivery system greater than 99%.Utilize lipofection amine 2000 (LF2000) and PEI (line style and branching) in contrast, study physical stability and anti-RNA enzymatic degradation with the siRNA complex of low-molecular weight chitoglycan preparation.In the report that great majority are sent about siRNA, adopt the cotransfection method that relevant target of non-physiology or irrelevant pDNA are mixed in the siRNA preparation.Therefore, our at first this influence of mixing of vitro examination in the cell line of stably express luciferase to the siRNA delivery efficiency by various polycations.Then, we have studied the prescription and the structure of siRNA preparation and low-molecular weight chitoglycan complex.More specifically say, studied effect, formulation parameter (charge ratio, siRNA concentration), the serum influence of low-molecular weight chitoglycan (chain length and degree of branching) structural change, and the reticent active efficient of these factors and outer-gene is associated.The cytotoxicity that has compared various siRNA preparations, for example external kinetics of luciferase genes silence.
These studies show that low-molecular weight chitoglycan might be used as the novel delivery system of siRNA (siRNA).Adopt polymine (PEI) and lipofection amine 2000 (LF2000) in contrast, make the chitosan and the siRNA formation complex of various chain lengths and check its physical stability and at the protective effect of enzymatic degradation.The cytotoxicity of in vitro study siRNA complex and luciferase genes are reticent active in 293 cells of stably express luciferase.Chitosan structure variation and formulation parameter have also been studied to the reticent active influence of siRNA complex luciferase.Low-molecular weight chitoglycan can be compounded to form physics stabilized nano grain (34-86nm) with siRNA, and the protective effect at the RNA enzymatic degradation is provided.The chitosan chain shows that than the higher higher importance of positive changes that causes of the charge ratio between long and/or low-molecular weight chitoglycan and the siRNA the reticent activity of external mediation luciferase is the highest.Different with PEI and LF2000, the siRNA complex of preparing with low-molecular weight chitoglycan keeps the reticent activity of its luciferase in the transfection media that contains 10% serum.Compare with LF2000 with PEI, the cytotoxicity of low-molecular weight chitoglycan is also minimum.Number-average degree of polymerization (DP n) be 85 monomeric unit (DP n85) the siRNA concentration that low-molecular weight chitoglycan requires to be low to moderate 44nM is expressed 95% silence to be implemented in the luciferase genes of keeping in the 293-Luc cell 5 days.Consider these factors simultaneously, our discovery shows that low-molecular weight chitoglycan is the effectively alternative delivery system that is used for siRNA.We past attempts report that the very short basic deacetylated low-molecular weight chitoglycan (18-34 monomeric unit) fully of chain length should be used for pDNA in vitro and in vivo and send.In present research, we have reported the structure-character relation of deacetylated substantially fully chitosan oligomer as the external delivery system of siRNA.For this reason, select the line style of various chain lengths and the chitosan oligomer of trisaccharide-replacement (branching).We find, send on the contrary with pDNA, and the above line style chitosan oligonucleotide of 34 monomeric units and siRNA form the complex of physically stable, and in HEK293 (293-Luc) cell of expressing luciferase the highest luciferase silence of external mediation activity.Obviously, the structure between siRNA and the pDNA-character relation is significantly different.This species diversity may be that the siRNA molecule is shorter because compare with pDNA, and pliability is lower and negative charge density is lower.Therefore, siRNA is compounded to form the stable and interionic interaction [18,33] that effectively nanoparticle need be stronger with polycation of physics.
In our research with the granularity (less than 100nm) of the siRNA complex of basic deacetylated fully low-molecular weight chitoglycan preparation unexpectedly more than the granularity little [33,34] of lipid, PLL and the high molecular weight chitosan (85% is deacetylated) of past report.This may be because of higher charge density on the complete substantially deacetylated chitosan main chain.In addition, raising of the dissolubility of low-molecular weight chitoglycan and viscosity reduction may be because the granularity little [35] of siRNA complex.Consistent with the report in past, siRNA-Luc nothing to do with pDNA (pGFP) causes significant luciferase reticent active [17] with term single gene bag (package) form cotransfection in external 293-Luc cell.Yet we find that active for gene silencing is significantly impaired when the siRNA preparation does not contain pDNA.Therefore, we think, pDNA, this high negative charge density macromolecular existence significantly promotion siRNA form the efficient of complex by the synergism improved and various polycation.
We also find, the high charge density of long chitosan chain and/or the complex that higher charge ratio not only will produce physically stable, and can also be at the efficient gene silencing of external acquisition.Our discovery is consistent with the result of the tree-shaped polymer of reporting in the past of PAMAM, the tree-shaped polymer complex of siRNA/ will obtain compound action and active for gene silencing preferably, just requires higher algebraically (generation number), higher charge ratio and higher siRNA concentration (100nM) [18].Preparation based on LF2000 is also recommended similar high concentration siRNA.In the present invention, the most effective low-molecular weight chitoglycan (line style DP n85) require siRNA concentration to be low to moderate 44nM, in the 293-Luc cell, to realize luciferase genes expression silencing greater than 95%.
And under the experiment condition of this research, the siRNA complex of preparing with long-chain line style low-molecular weight chitoglycan keeps the reticent activity of its luciferase in the transfection media that contains 10% serum.Reticent activity is retained most probable reason and is, and is opposite with PEI and LF2000, and these complex are anti-the gathering in this higher relatively serum-concentration, reflected enhanced colloidal stability.Find that chitosan oligomer short and branching has lower active for gene silencing, possible reason is that the sterically hindered siRNA that causes of charge interaction is compound impaired between charge density reduction and chitosan main chain and the siRNA.
Consistent with the cytotoxicity minimum with the siRNA complex of high molecular weight chitosan preparation of report, we find even adopt high siRNA concentration (150nM) in the preparation, to use DP in this research nThe 293-Luc cell keeps dehydrogenase activity [33] in its born of the same parents after the 85 low-molecular weight chitoglycan transfections.The cytotoxicity of low-molecular weight chitoglycan utilizes the siRNA concentration of 50-100nM can cause cell viability significantly to reduce (60-50%) far below the tree-shaped polymer gained of disclosed PAMAM of past result in the tree-shaped polymer of PAMAM.
At last, with the PEI[17 of past report] compare, with the siRNA complex onset of line style low-molecular weight chitoglycan preparation early and to keep luciferase reticent active.Estimate DP nThe dynamic (dynamical) improvement of 85 chitosan oligonucleotide be the cellular uptake of little nanoscale siRNA complex improve and complete siRNA born of the same parents in continue the result that discharges.
2. material and method
2.1. material
The GMP-level plasmid (gWiz that contains cytomegalovirus promoter and Lampyridea luciferase (pLuc) or green fluorescent protein (pGFP) TM) available from the love moral of North Dakota method dagger-axe hear company (Aldevron, Fargo, ND, USA).Lipofection amine 2000 (LF2000) is available from hero company (Invitrogen).Line style PEI; ExGen 500 (molecular weight 22kDa) is available from German Fu Laimengta company (Ferementas).Branching PEI (molecular weight 25kDa) available from the Sweden A Erde Ritchie company of Stockholm, SWE (AldrichSweden, Stockholm, Sweden).
2.2.siRNA duplex
SEQ ID NO.1:siGL3 (justice, 5 '-CUUACGCUGAGUACUUCGAdTdT-3 ';
SEQ ID NO.2: antisense, 5 '-UCGAAGUACUCAGCGUAAGdTdT-3 ') be the siRNA duplex of the unmodified of targeting luciferase genes (siRNA-Luc), by medical probe company (MedProbe, Lund, Sweden) customization [28] of the blue moral of Sweden.
SEQ ID NO.3: mispairing siRNA; The non-targeting siRNA#1 of siCONTROL (siCON1; Justice, 5 '-UAGCGACUAAACACAUCAAUU-3 ';
SEQ ID NO.4: antisense, 5 '-UUGAUGUGUUUAGUCGCUAUU-3 ') by the DR company limited of state of Colorado Lafayette (Dharmacon Research, Inc., Lafayette, CO) customization.
2.3. low-molecular weight chitoglycan
Deacetylated (degree of deacetylation＞99.8% of complete N-that has prepared various chain lengths; FA＜0.001) low-molecular weight chitoglycan (branching chitosan) that line style and 7% trisaccharide replace and according to described the sign [25,29].All adopt number-average degree of polymerization (DP n) be the low-molecular weight chitoglycan of 34,50 and 85 monomeric units.Size exclusion chromatograph (SEC-MALLS) with multiple angle laser light scattering is analyzed chain length distribution.
2.4. cell
Human embryonic kidney cell line HEK 293 (293 cell) derives from Maryland, USA Rockville (Rockville, MD, ATCC USA).The HEK 293 (293-Luc cell) of the stably express luciferase of expression Lampyridea luciferase is by parfe doctor Hao Kasiji (the Dr.Paavo Honkakoski of pharmaceutical college of Kuopio, Finland university, Department of Pharmaceutics, University of Kuopio Finland) gives [30].The ovarian cancer cell line of stably express luciferase (SKOV-3-Luc) is Archie ENI doctor (the Dr.Achim Aigner by Marburg, Germany Philips university's pharmacology and toxicity institute, Department of Pharmacology and Toxicology, Philipps-University Marburg Germany) gives [17].All cells is cultivated according to supplier's suggestion.
2.5.siRNA the preparation of complex
Chitosan is dissolved in the MilliQ sterilized water of pH6.2 and carries out aseptic filtration and prepare chitosan storing solution (0.2 mg/ml).At vortex mixed machine (Heidolph REAX 2000,4 grades, the triumphant uncle of this handkerchief song of Sweden laboratory (Kebo Lab, Spanga, when Sweden)) going up vigorous stirring, with in chitosan and siRNA storing solution or siRNA/pDNA (under the situation of cotransfection) the adding MilliQ sterilized water, the preparation chitosan complexes is as described in [25] successively.Every microgram pDNA or siRNA adopt the chitosan of following various content prepared charge ratio 1: 1 chitosan complexes of (+/-): 0.58 microgram line style chitosan, the chitosan that 0.69 microgram is replaced by 7%A-A-M (branching chitosan) [25,27].In the vigorous stirring, PEI solution is added among siRNA storing solution or the siRNA/pDNA (under the situation of cotransfection) on the vortex mixed machine, the siRNA complex of preparation PEI is as described in [31].Said preparation at room temperature left standstill carry out transfection after about 10 minutes.For forming the LF2000 complex, on the vortex mixed machine, in the vigorous stirring siRNA storing solution or siRNA/pDNA (under the situation of cotransfection) are added in the chitosan solution.With transfection medium OptiMEM I dilution siRNA and LF2000 solution.The LF2000 complex at room temperature left standstill carry out transfection after about 30 minutes.According to basic test, all select and the LF2000 complex (data not shown) of used charge ratio 15: the 1 PEI complex of (+/-) and weight ratio 2: 1 (+/-).
2.6. gel retardation assay
Utilize the physical stability of agarose gel blockade test research siRNA complex.4% agarose of preparing in the employing 40mM TAE buffer (
Figure A20078003434400121
Agarose, and the blue company limited of the CBS Roc of Massachusetts, United States Roc orchid (Cambrex Bio Science Rockland, Inc., Rockland, ME, USA), as described in [25].(protective effect that siRNA that research after 30-90 minute forms complex resists enzymatic degradation is hatched in Britain An Bi company (Ambion, UK)), as described in [18] with 1.5U RNA enzyme A.After hatching, make complex dissociation, check the integrity of siRNA with the agarose blockade test with heparin (5 mg/ml).The siRNA that obtains from storing solution in contrast.
2.7. the size measurement of chitosan poly complex (polyplex)
The size of complex adopts nanometer screening instrument ZS, and ((Malvem Instruments, Malvern UK), measure by photon correlation spectroscope, as described in [25] in the bridle literary composition equipment company of Britain's bridle literary composition.Prepare the complex that siRNA concentration is 5 mcg/ml with MilliQ water.All measurements are carried out under 25 ℃.
2.8. external infection test
Transfection was tested preceding 24 hours, and (293,293-Luc) (in the Coase tower company of Britain Camb (Costar, Cambridge, UK)), transfection reached the 80-90% cell and converge the same day to 96-hole tissue culturing plate for cell and SKOV-3-Luc cell inoculation with HEK 293.PH 7.4 serum-free mediums (culture medium that OptiMEMI serum reduces, Sweden wear than Ji Buke/BRL life sciences AB (Gibco/BRL LifeTechnologies AB, Taby, Sweden)) in or in the presence of 10% serum (FBS), carry out transfection.(300mOsm/kg) oozed in adding mannitol realization etc.With the OptiMEM washed cell of preheating, and in each hole, add 50 microlitre siRNA complex formulations.In cotransfection experiments, add 0.33 microgram pDNA (pLuc or pGFP) in every hole.Comprise mispairing (contrast) siRNA in all experiment in vitro.After hatching in 5 hours, remove preparation, add 0.2 milliliter of fresh medium.For super experiment two days later, each second day replacing culture fluid.24-120 hour predetermined point of time after transfection, with PBS (pH7.4) washed cell of preheating, and with luciferase lysis buffer (the Pu Luomaige company of state of Wisconsin Madison (Promega, Madison, WI)) cell lysis.(the Midi tower company in Australian Vienna (Mediators PhL, Vienna, Austria)) measures luciferase genes and expresses to use luminometer then.(standard curve of the Sigma company of St. Louis, the Missouri State (Sigma, St Louis, MO)) preparation is determined the expression of luciferase by the Lampyridea luciferase.
2.9. dehydrogenase activity (MTT method) in the born of the same parents
By the MTT method estimate various siRNA preparations in the 293-Luc cell to born of the same parents in the influence (Cytotoxic measurement) of dehydrogenase activity, as described in [32].In brief, transfection 293-Luc cell as mentioned above.After the transfection 5 hours, and the MTT (bromination 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium) that adding is prepared with phosphate buffered saline (PBS) (PBS) (the Sigma company of German Di Senhaofu (Sigma, Deisenhofen, Germany)) solution.After 4 hours, add 100 microlitre acid-isopropyl alcohols (the 0.04M hydrochloric acid of preparation) dissolving first
Figure A20078003434400131
Crystallization.Plate instrument (TA company limited (the TECAN Satire of Australian Ge Laodi is read in employing 2, Tecan Austria GmbH, Grodig, Austria)) and measure the absorbance of 570nm, carry out background correction at 690nm.The absorbance of the culture medium that equipment will be handled in the same manner is set at 0.The interior dehydrogenase activity of born of the same parents of handling cell is associated with contrast (being untreated) cell, and is calculated by following equation:
Dehydrogenase activity=[A (test) * 100/A (contrast)] in the relative born of the same parents of %,
Wherein A (test) and A (contrast) are respectively the absorbances of handling cell and control cells.Impose a condition down at another, cell was grown in culture medium 24 hours.Then, with MTT agent treated cell to detect the delayed toxicity of various preparations.
2.10. data analysis
Test utilizes quadruplicate sample to carry out at least twice.All data are expressed as meansigma methods ± standard deviation.With the significant difference between the ANOVA analysis meansigma methods.P＜0.05 o'clock thinks that the average differences is remarkable between group.
Physical stability and enzyme protection effect
We have studied the line style of various chain lengths and the ability of branching chitosan oligomer and siRNA formation stable compound first in gel retardation assay.Have only the complex of preparing with the long line style oligonucleotide of higher charge ratio to keep siRNA at pH8.0, this pH is electrophoretic buffer pH (Figure 1A) commonly used.Number-average degree of polymerization (DP n) be 85 monomeric unit (DP n85) physical stability that line style low-molecular weight chitoglycan provides under the charge ratio of all detections is the highest.On the contrary, when the pH of gel buffer liquid reduced to 7.4, the low-molecular weight chitoglycan of all detections can form stabilized complex with siRNA.Branching or the physical stability reduction prompting of the siRNA preparation of short chitosan oligomer under the pH value that raises, for forming the complex of comparing physically stable with the pDNA complex that the past is optimized with the low-molecular weight chitoglycan preparation, polycation charge density of having relatively high expectations and stronger siRNA interaction [25,27].
Because enzymatic degradation may be the restrictive factor of active for gene silencing, we have also studied the ability (incubation time adopted 30-90 minute) that selected low-molecular weight chitoglycan protection siRNA avoids RNA enzyme A enzymatic degradation.
According to the requirement of gene delivery, to compare with naked siRNA, the low-molecular weight chitoglycan of all detections and positive control (PEI and LF2000) provide the protective effect of antagonism enzymatic degradation, and naked siRNA is by RNA enzyme A degrade fully (Figure 1B).Need carry out long incubation time (2 hours) with heparin and use line style DP to destroy n85 and the complex of LF2000 preparation, reflect that comparing physical stability with the polycation of other detections improves (data not shown goes out).
Granularity
Because the granularity of siRNA preparation can influence its tissue distribution and cellular uptake greatly, so we have studied the granularity with the siRNA of low-molecular weight chitoglycan preparation.Table 1 shows that low-molecular weight chitoglycan and siRNA self assembly form nanorize particle (34-86nm).The granularity of gained particle depend on component+/-charge ratio.Though under the situation of minimum charge ratio 10: 1 (+/-), obtain small grain size (34-46nm), use higher charge than causing relatively large granularity (61-86nm).Granularity is measured by photon correlation spectroscope.
Table 1
A/P?10 A/P?30 A/P?60
DPn18 38.1±1.2 54.5±0.1 62.5±3.7
DPn34 37.7±0.5 53.1±0.9 68.2±3.0
DPn50 34.0±0.5 51.7±1.2 63.6±1.1
DPn85 37.4±1.1 51.6±3.3 61.0±2.5
DPn34-AAM-7％ 38.5±0.8 50.8±0.4 68.9±2.7
DPn85-AAM-7％ 46.0±2.5 69.9±1.7 86.1±2.9
The silence that compares cotransfection and stably express target
Because in most of in vitro testses, siRNA molecule and its pDNA target are sent (cotransfection) simultaneously, we utilize mispairing (non-silence) siRNA in contrast, have detected the efficient that low-molecular weight chitoglycan is sent pDNA (pLuc) with the gene bag of the siRNA (siRNA-Luc) of targeting same report sequence of coding Lampyridea luciferase report sequence first.In pDNA dosage, our laboratory, send under the optimization transfection conditions of the most effective low-molecular weight chitoglycan optimized and charge ratio [27], in 293 cells (not expressing luciferase report sequence), carry out transfection for pDNA.By branching DP nThe siRNA-Luc that 34 chitosan oligomer or LF2000 send causes comparing with control formulation (only pLuc), and luciferase expression is significantly struck and subtracted (Fig. 2 A).For two kinds of delivery systems, all do not observe mispairing siRNA and significantly suppress luciferase expression.
Yet, when siRNA-Luc under the same conditions changes into when only being delivered to the 293-Luc cell (using more relevant situation with gene silencing) of stably express luciferase by selected polycation, the reticent activity of gained luciferase is very low, realize the siRNA concentration (Fig. 2 B) that the significance silence of luciferase expression is had relatively high expectations.
For whether research cotransfection technology can influence active for gene silencing in the 293-Luc cell, the plasmid (pGFP) that will have nothing to do mixes in the identical siRNA-Luc preparation of above-mentioned detection.Similar with 293 cell gained results, branching chitosan oligomer, PEI and LF2000 strike with the significant luciferase expression of minimum siRNA concentration (1-30 nanogram/hole is equivalent to 1.5-40 nanomole/hole) mediation and subtract (40-85%) (Fig. 2 C).In the siRNA preparation, mix pDNA (cotransfection) active for gene silencing is had positive effect.These results suggest, be used for preparation that siRNA sends require different with pDNA, for successfully sending siRNA by polycation and need characterize important parameters external.
The structural change of low-molecular weight chitoglycan and formulation parameter are to the influence of active for gene silencing
A) chain length, main chain degree of branching and charge ratio
For the influence of checking that chitosan structure and formulation parameter are sent siRNA to low-molecular weight chitoglycan, we have at first tested the influence to siRNA complex external mediation luciferase expression silence in the 293-Luc cell of chain length, degree of branching and charge ratio.For the line style chitosan, chain length does not rely on the remarkable luciferase silence (Fig. 3 A) of charge ratio greater than the chitosan mediation of 34 monomeric units.Compound-mediated luciferase silence with the preparation of branching chitosan oligomer depends on charge ratio and chain length (Fig. 3 B) simultaneously.For long branching chitosan oligomer, higher charge ratio can remedy the negative effect of chitosan main chain branching (replacement).These results highlight, the high charge density of low-molecular weight chitoglycan not only can produce the complex of physically stable, and can be provided at external effective siRNA preparation.
B) the siRNA concentration and the relative efficiency of line style low-molecular weight chitoglycan
Next step, research siRNA concentration is to the active influence of luciferase silence with the complex of various line style low-molecular weight chitoglycans (with constant charge ratio) preparation in the 293-Luc cell.For minimum siRNA concentration (15-50 nanogram/hole is equivalent to 22-73 nanomole/hole), line style DP n85 complex strike luciferase expression and subtract 72-95% and show the most high-effect (Fig. 4) with respect to the contrast untreated cell.When siRNA concentration when 70 increase to 300 nanograms/hole (103-440 nanomole/hole), remove DP nOutside 18, the line style chitosan complexes of being tested shows the reticent curve of suitable luciferase.With line style, in realizing the reticent activity of high gene, effect is arranged than the physical stability of the higher support of the usefulness siRNA preparation of the complex of long-chain low-molecular weight chitoglycan preparation.
In-vitro transfection under serum exists
We have also studied the influence of the serum in the transfection medium to various siRNA preparation efficient.In the presence of 10% serum, be the line style chitosan and the branching DP of 34-85 monomeric unit with the DP value nThe siRNA complex of 85 chitosan oligomer preparation keeps its active for gene silencing (Fig. 5) in the 293-Luc cell.)。On the contrary, the active for gene silencing of the siRNA complex of usefulness PEI and LF2000 preparation is impaired.The impaired a kind of possible reason of efficient is a particle accumulation during the transfection.
Vitro cytotoxicity
For guaranteeing that the reticent efficient of higher luciferase is not the result that cytotoxicity raises, we adopt the MTT method to study the various polycation complex pair cell forms of siRNA (150 nanomole) preparation with relative higher concentration and the influence of the interior dehydrogenase activity of born of the same parents.After the transfection 5 hours, do not observe and use line style DP nDehydrogenase activity (Cytotoxic measurement) influential (Fig. 6) in the siRNA complex pair cell form of 85 preparations and the born of the same parents.On the contrary, observe significant toxic effect with PEI and LF2000, wherein line style PEI toxicity is the highest.Though the cell of handling with the LF2000 complex recovered dehydrogenase activity in its born of the same parents after 24 hours, the further reduction of cell demonstration dehydrogenase activity after 24 hours of handling with PEI.These results show, even use the siRNA of higher concentration, line style DP nThe acute cytotoxicity of 85 low-molecular weight chitoglycans also is lower than PEI and LF2000.
The external kinetics of siRNA
At last, studied the time-histories that siRNA-Luc is delivered to luciferase silence behind the 293-Luc cell.Tested low-molecular weight chitoglycan, PEI and the LF2000 of DP value for 34-85 monomeric unit.Do not comprise DP n18 chitosans are because it is not as long-chain chitosan effective (Fig. 4).Use line style DP nThe reticent onset of the compound-mediated luciferase of the siRNA of 85 preparations early detects remarkable effect (70%) after 1 day, reach maximum (92%) (Fig. 7 A) after 2 days.Active for gene silencing continues 5 days, points out stable release in the complete siRNA born of the same parents.The reticent aerodynamic efficiency of the luciferase of branching chitosan oligomer is not as line style oligomer (Fig. 7 B).The gene silencing effect of LF2000 and PEI is than the effect low (Fig. 7 C) of low-molecular weight chitoglycan mediation.In another kind of cell line SKOV-3-Luc cell, the reticent kinetics of luciferase provides the result (Fig. 7 D) who is similar to the 293-Luc cell.
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Claims (19)

1. compositions, it comprises the complex of following composition:
(a) degree of polymerization (DP n) be the low-molecular weight chitoglycan of 30-300, the degree of deacetylation of wherein said low-molecular weight chitoglycan is greater than 90%; With
(b) oligonucleotide.
2. compositions as claimed in claim 1 is characterized in that, described low-molecular weight chitoglycan utilizes chemistry or enzyme method to be obtained by high molecular weight chitosan.
3. compositions as claimed in claim 1 is characterized in that the degree of deacetylation of described low-molecular weight chitoglycan is greater than 95%.
4. compositions as claimed in claim 3 is characterized in that the degree of deacetylation of described low-molecular weight chitoglycan is greater than 99%.
5. compositions as claimed in claim 1 is characterized in that, described compositions has clean positive charge ratio basically.
6. compositions as claimed in claim 1 is characterized in that, with targeting aglucon and the stabilizing agent described low-molecular weight chitoglycan of deriving.
7. compositions as claimed in claim 1 is characterized in that, described oligonucleotide comprises the reticent sequence of expressing its function when introducing host cell.
8. compositions as claimed in claim 7 is characterized in that, described oligonucleotide is selected from: RNA molecule, antisense molecule, ribozyme and Microrna.
9. compositions as claimed in claim 8 is characterized in that, the pH scope of described compositions is 3.5-8.0.
10. compositions as claimed in claim 9 is characterized in that, the pH scope of described compositions is 7.1-7.6.
11. one kind prepares method for compositions according to claim 1, said method comprising the steps of:
(a) low-molecular weight chitoglycan is contacted with aqueous solvent;
(b) aqueous solution and the oligonucleotide of blend step (a) in aqueous solvent; With
(c) pH that keeps compositions is 3.5-8.0.
12. method as claimed in claim 11 is characterized in that, described method comprises that also the volume of the product solution that reduces step (b) generation is to obtain required composition concentration.
13. give mammiferous method with nucleic acid, described method comprises introduces mammal with compositions as claimed in claim 1.
14. method as claimed in claim 13 is characterized in that, by pulmonary, nasal cavity, oral, eyes, buccal, Sublingual, part, rectum or vaginal approach gives mucosal tissue and described compositions is introduced in the mammalian body.
15. method as claimed in claim 13, it is characterized in that, by in intravenous, intramuscular, intradermal, intracranial, the canalis spinalis, subcutaneous or intracardiac parenteral approach gives submucous tissue, or give other body surfaces of exposing during internal organs, blood vessel or the surgical operation or body cavity and described compositions introduced mammal.
16. method as claimed in claim 13 is characterized in that, described compositions is the oligonucleotide that can express its function at least a mammalian cell.
17. one kind is used method for compositions according to claim 1, described method comprises that manufacturing is used for preventative or the mammiferous medicine of therapeutic treatment, and described medicine comprises compositions as claimed in claim 1.
18. one kind is used according to claim 1 compositions as the method for diagnostic reagent, described method comprise in vivo or the in-vitro diagnosis method in use.
19. compositions as claimed in claim 17 is used for application in the gene therapy, antisense therapy of preventative or therapeutic treatment malignant tumor, autoimmune disease, hereditary disease, pathogen infection and other pathologic conditions or the medicine that the genetic vaccine inoculation is used in manufacturing, described application comprises by following route of administration and gives described compositions is introduced in the mammalian body:
A) give mucosal tissue by pulmonary, nasal cavity, oral, eyes, buccal, Sublingual, rectum or vaginal approach,
B) by in intravenous, intramuscular, intradermal, intracranial, the canalis spinalis, the subcutaneous or intracardiac parenteral approach that gives gives submucous tissue,
C) give other body surfaces or the body cavity that expose during internal organs, blood vessel or the surgical operation.
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